Using human hybridoma technology, we have generated eleven new monoclonal antibodies to rabies virus and autoantigens. These antibodies can be classified into either monoreactive high-affinity or polyreactive low- affinity molecules. One of these antibodies has rabies virus neutralizing activity. Because of the instability of human hybridomas, we have rescued and cloned Fab genes from several antibodies and expressed these genes in E. coli. The bacteria-expressed recombinant Fabs retained their binding specificity. Since the immunoglobulin genes are cloned in bacterial vectors, they can be molecularly manipulated in order to study the structure-function relationship of an antibody molecule. In this regard, we have used mAb 57 as a model antibody to investigate the effects of somatic mutations on its biological functions. Our results showed that a single point mutation in the complementarity- determining region (CDR)-1 can lead to a significant loss of binding activity. Several mutant antibodies are being used for further immunological and biochemical studies. The biological functions as well as the molecular mechanisms by which polyreactive antibodies interact with many dissimilar antigens have not been determined. In this study, our results showed that CDR-3 of polyreactive antibodies is slightly longer. It has more arginine and aromatic amino acid residues than that of the monoreactive antibodies. Based on this knowledge, we are in the progress of using a PCR-based site-specific mutagenesis method to replace these residues with aliphatic and uncharged amino acids in order to determine whether polyreactivity will be retained. To further investigate the role of CDR- 3 in polyreactivity, we plan to generate chimeric antibody molecules containing CDR-3 from polyreactive antibodies that has been grafted onto a monoreactive antibody backbone and assay for their binding specificity.